Structure, Activity and Dynamics of Human Serum Albumin in a

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Structure, Activity and Dynamics of Human Serum Albumin in a Crowded Pluronic F127 Hydrogel Atanu Nandy, Subhajit Chakraborty, Somen Nandi, Kankan Bhattacharyya, and Saptarshi Mukherjee J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b00219 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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The Journal of Physical Chemistry

Structure, Activity and Dynamics of Human Serum Albumin in a Crowded Pluronic F127 Hydrogel Atanu Nandy,1 Subhajit Chakraborty,1 Somen Nandi,2 Kankan Bhattacharyya1* and Saptarshi Mukherjee1* 1Department

of Chemistry, Indian Institute of Science Education and Research Bhopal,

Bhopal Bypass Road, Bhopal 462 066, Madhya Pradesh, India 2School

of Chemical Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India

Abstract Structure, activity and dynamics of a plasma protein, human serum albumin (HSA), inside a crowded environment of F127 gel are studied by circular dichroism (CD), fluorescence correlation spectroscopy (FCS) and picosecond time-resolved fluorescence spectroscopy. For our purpose, the protein is covalently labeled by a maleimide dye, CPM [7-(diethylamino)-3-(4maleimidylphenyl)-4-methyl-coumarin]. The circular dichroism (CD) spectra suggest that the protein remains more structured in the gel reflecting about the biological activities of the protein. FCS results demonstrate that compared to bulk water (buffer solution) translational diffusion is about 59 times slower inside F127 gel. This indicates higher translational friction (viscosity) sensed by the probe (CPM). On the contrary, rotational relaxation (and hence, rotational frictions) is more or less similar in F127 gel and in bulk water. FCS results further indicate that the time-scales of conformational relaxation of the protein are substantially slowed down inside the crowded environment of F127 gel. The fast component of conformational relaxation is retarded by 55 times and the slow component by 20 times. Fluorescence spectra of CPM bound to HSA show a 5 nm red shift in the emission maximum implying that the microenvironment of the probe, CPM, attached to the protein is more polar inside the gel. Solvation dynamics study reveals a faster relaxation for CPM labeled HSA inside the gel ( ~ 300 ps) compared to that of the protein in bulk water ( ~ 600 ps).

*E-mail:

[email protected], [email protected]

1

ACS Paragon Plus Environment

Fax No: +91-755-409-2392

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.

Introduction A protein carries out its biological activities in the extremely crowded environment inside

a cell. 30% of the volume of a cell is occupied by large biological macromolecules at a very high concentration (several hundred gL-1).1-7 There is a vigorous recent interest to understand how crowding affects gene expression,4 protein folding and stabilization,5-7 conformational transition of macromolecules etc.8-9 Many groups have examined the effect of polymers on protein in aqueous solutions by experiments,10-21 and by computer simulations as well.22-24 Using single molecule photon stamping FRET trajectory analysis, Lu and co-workers studied the effect of a relatively inert polysaccharide, Ficoll 70 (a highly-branched copolymer of sucrose and epichlorohydrin, molecular weight ~70 kD having radius 5.5 nm), on the folding of a calcium sensing protein (calmodulin).10 Gruebele and co-workers investigated steric and non-steric interactions in vitro by the crowding agent, Ficoll 70, to understand the crowding effect inside a cell.11,12 Pielak and co-workers studied the effect of reconstituted cytoplasm on the stability of a protein.13 Fluorescence correlation spectroscopy (FCS) has been used to monitor the effect of added polymer (polyethylene glycol) on the size of a protein in an aqueous solution.15-17 Chowdhury and co-workers studied FRET and solvation dynamics in a protein in the presence of several crowding agents such as, dextran, polyethylene glycol and another protein.18,19 These experiments are intermediate in complexity between studying an isolated protein in a solution6-7 and that in a single live cell studied by super-resolution microscopy and time-resolved confocal microscopy.25,26 In general, it is believed that crowding and associated excluded volume effect accelerates protein folding, aggregation and enhances stability and modulates enzyme activity. In the present work, we focus on the structure and dynamics of a protein in a macroscopically rigid but microscopically porous polymer hydrogel.27-30 The triblock copolymer, pluronic F127 with the formula (polyethylene oxide; PEO)100 – (polypropylene oxide; PPO)70 – (PEO)100 forms a micelle at a low concentration and a gel at high concentration.31-35 According to SANS studies, in these gels, spherical micelles of diameter 24 nm remain immobilized (Scheme 1A).31-34 Close packing of such spherical micelles leave about 50% void space. Such a structure is highly heterogeneous where local polarity and viscosity varies rapidly over a distance of 12 nm (radius of the spherical micelles). The “void” region is water like with high polarity and very low viscosity. The PPO core is highly nonpolar and is of very high viscosity. The corona region containing PEO chains is hydrophilic and involves water penetration core with a viscosity intermediate between the core and the void. Previous studies 2


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suggest that one can estimate viscosity of different regions of this immobilized spherical micelles in a gel using different fluorescent probes with varying hydrophobicity which localize in different regions (core and corona) of the gel.36-37 Different regions of this immobilized micelles have been probed earlier by fluorescence anisotropy decay36 and by FCS.37 It has been shown earlier that the inner hydrophobic PPO core (reported by hydrophobic DCM probe molecule) has a viscosity (i.e. translational friction) about 14 times higher than that at the hydrophilic corona (PEO) region (reported by charged C343 probe).37 Compared to bulk water, viscosity is 300 times more in the core and 40 times higher at the corona.37

(A) Hydrophilic corona (PEO block) Pore (“Void”) Hydrophobic core (PPO block)

(B)

O O

H

N

N

N

S

O

O

O

HS N

O

Free CPM, Non-fluorescent

O

O

Bound CPM, Fluorescent

Scheme 1. (A) Schematic representation of micelle and gel formed by a triblock copolymer Pluronic F127; (B) Covalent labeling scheme of thiol group (-SH) of protein by CPM probe. In order to investigate the rotational relaxation, translational diffusion and picosecond solvation dynamics, we have covalently labeled the lone cysteine residue (Cys-34) of human serum albumin (HSA) by a coumarin maleimide probe, CPM, [7-(diethylamino)-3-(43



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maleimidylphenyl)-4-methylcoumarin, Scheme 1B].38-40 We have shown that the components of conformational relaxation as monitored using FCS is slowed down significantly in the gel matrix. On the other hand, the average time-scale of fluorescence anisotropy decay is marginally affected in the gel while translational diffusion undergoes appreciable retardation in F127. Interestingly, Solvation dynamics studies reveal a faster relaxation of the solvent response around CPM covalently attached to HSA inside the gel pores as compared to buffer. 2.

Experimental Section

2.1

Materials Human

Serum

Albumin

(HSA),

pluronic

triblock

copolymer

F127,

sodium

monophosphate and sodium diphosphate were purchased from Sigma Aldrich and used without further purification. Laser grade dye, CPM [7-(diethylamino)-3-(4-maleimidylphenyl)-4methylcoumarin] was purchased from Exciton and used as received. 50 mM phosphate buffer was prepared with sodium monophosphate and sodium diphosphate maintaining the pH 7.4. F127 exists in the gel phase in 30 wt% aqueous solution at 22 °C temperature.31-35 All the measurements were carried out at this temperature. 2.2

Methods

A.

Labeling of HSA by CPM dye Human Serum Albumin (HSA) contains one free cysteine amino acid residue at 34

position (Cys-34) in domain IA that can be labeled covalently by CPM dye through Micheal addition reaction (Scheme 1B). For this, we have followed the procedure as described earlier with minor modification.38-40 The concentration of labeled protein was determined by following the procedure as described earlier, and it was measured to be 5.5 μM by optical absorbance. The labeling efficiency was determined spectro-photometrically to be 80%. B.

Steady-state Absorption and Fluorescence Spectra Steady-state absorption spectra were acquired on a Cary 100 UV-Vis spectrophotometer

with the baseline correction and fluorescence emission spectra were recorded in a Fluorolog 3111 spectrofluorimeter using a quartz cuvette. All the data were recorded with the correction of background signal. The protein solution was excited at 295 nm to eliminate the contribution of fluorescence emission coming from the tyrosine residue of the protein. 4


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C.

Circular Dichroism (CD) Measurements Circular dichroism (CD) measurements were carried out using a quartz cuvette of 2 mm

path length in a JASCO J-815 spectropolarimeter. Baseline correction was done before recording any CD data and average CD data were taken from two successive scans recorded with a scan rate 50 nm min-1. D.

Confocal Microscope Set-up for FCS Study FCS experiments of the CPM labeled HSA (~10 nM) in bulk solution (buffer) and inside

the F127 gel were performed in a confocal setup (PicoQuant, MicroTime 200) with an inverted optical microscope (Olympus IX-71), described in detail elsewhere.37,39,40 Briefly, the fluorophore was excited at λex = 405 nm and fluorescence is separated from the exciting laser by a dichroic mirror (405DCXR, Chroma) and a suitable band-pass filter (HQ425lp, Chroma). The fluorescence was focused through a pinhole (50 μm) onto a 50/50 beam splitter (Chroma) and then allowed to strike two MPD (micro photon device) detectors. The signal was subsequently processed by the PicoHarp-300 time-correlated single photon counting card (PicoQuant) to generate the autocorrelation function, G(τ). The data were collected in time-tagged time-resolved (TTTR) mode. The autocorrelation function is very sensitive to the collar settings of the objective of the microscope. We fixed the objective collar setting to 0.17 for each experiment. E.

Picosecond Time-resolved Fluorescence Decays Time correlated single photon counting (TCSPC) technique was used for the recording of

fluorescence decays at different emission wavelengths for solvation dynamics and FRET studies. For excitation at 405 nm, we used a picosecond diode laser (IBH NanoLEDs) with pulse width 100 ps. For excitation at 295 nm, we used a LED source with pulse width 800 ps. The samples were thus excited at specific wavelengths (405 nm for CPM and 295 nm for tryptophan residue of the HSA) and the fluorescence signals were collected using a Hamamatsu MCP photomultiplier (model R-3809U50) at the magic angle polarization of 54.75°. The typical FWHM (full width at half maxima) value of the temporal responses from the instrument using a liquid scatterer (ludox solution) are found to be 100 ps for 405 nm and 800 ps for 295 nm. The fluorescence decays were deconvoluted using the instrument response function (IRF) of the detector. 5



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For fluorescence anisotropy experiment, the analyzer was rotated at regular intervals in order to obtain parallel (I‖) and perpendicularly (I⊥) polarized fluorescence. The anisotropy function r(t) was expressed as

r(t) 

I (t)  GI  (t)

(1)

I (t)  2GI  (t)

The G value was determined by tail matching of I║ and I⊥ components of fluorescence intensity of a probe whose rotational relaxation is very fast, (e.g. coumarin 102 in methanol)40 and was found to be 1.2. All the fluorescence decays have been fitted using DAS-6 decay analysis software. F.

Analysis of FCS traces In FCS, the autocorrelation function G( ) of fluorescence intensity is defined as39

G τ 

 δF  0  δF  τ  

(2)

 F 2

where, F(0) and F(τ) denote the fluorescence intensity at time 0 and at a lag time τ around the mean value, respectively. For CPM labeled HSA in bulk water, we had previously analyzed the FCS data to three different models and the best fitting was obtained for the 3D model involving single component diffusion and two components of relaxation.39 In the present work we used similar model for fitting the FCS data (Equation 3). 1

1 τ   τ  G AC τ   1   1  2  N  τD   ω τD 

1/2

τ τ    {1  A 1 e τ R1  A 2 e τ R2  A 1  A 2 }{1  A 1  A 2 } 1  (3)  

In this equation, N is the total average number of fluorophore molecules in the observed volume. τD is the diffusion time of the diffusing species, ω is the height-to-diameter ratio of the 3D Gaussian confocal volume and defined as ω = ωz/ωxy, where ωz is the longitudinal radius and ωxy is the transverse radius of the observation volume. τRi is the relaxation time for an exponential component with an associated amplitude of Ai.

6


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We calibrated ω using standard R6G solution in water with the known diffusion coefficient (Dt = 426 m2s-1)41 and found to be 5. ωxy is observed to be 270 nm for 50 μm pinhole. The confocal volume of our microscope setup is found to be 0.55 fL. Data have been analyzed with the IgorPro 6 software. Relaxation components of CPM-HSA do not exhibit any dependence on excitation power.39 We have varied the observation volume by using four different sizes of the pinhole (50, 75, 100 and 150 µm). D increases monotonically with increase in pinhole size with no systematic change in the values of relaxation times (Figure S1 and S2, ESI). Diffusion coefficient (Dt) is calculated from the value of transverse radius of the confocal volume (ωxy) and diffusion time (τD) using the following equation,

Dt  G.

ω xy 2

(4)

4τ D

Analysis of Solvation Dynamics The time resolved emission spectra (TRES) of CPM covalently linked to HSA were

constructed from the steady-state emission spectra and the parameters obtained from the best fit of the picosecond fluorescence transients following the procedure proposed by Maroncelli and Fleming.42,43 The solvation dynamics is described by the decay of the solvent correlation function C(t), defined as,

C(t) 

 (t)   ( )  (0)   ( )

(5)

where, (0), (t) and (∞) are the emission frequencies at time 0, t and ∞, respectively. The decay of the solvent correlation functions, C(t), were fitted to a single or double-exponential decay.

 C(t)   a i e

t τi

(6)

The temporal response of the picosecond setup is 100 ps as mentioned above. Therefore, it is very clear that a large part of ultrafast component of solvation dynamics taking place in a time-scale of shorter than ~100 ps will be certainly missed by this set-up. The 7



Page 8 of 32

solvation missed has been obtained according to the procedure proposed by Fee and Maroncelli.43 The following equation has been used to calculate the solvation missed and the true emission frequency at time zero, ν theo 0 



np ν theo 0  ν system  ν np abs abs  ν em



(7)

np np where, ν theo 0  indicates theoretical fluorescence maximum at zero time. ν abs and ν em

represent steady-state absorption and fluorescence maxima of the probe in a non-polar solvent, respectively. ν system denotes the absorption maximum of the system. In our study, the percentage abs of ultrafast component of solvation missed has been calculated by using CPM labeled cysteine as reported earlier.40 The absorption and emission maxima of CPM labeled cysteine in non-polar solvent (1,4-dioxane) are taken as 391 nm and 451 nm, respectively.40 From this, we have calculated the percentage missed of solvation for CPM labeled HSA in buffer solution as well as inside the F127 gel. H.

Esterase Activity: We have studied the biological activity (esterase activity) of the

labeled and unlabeled protein (HSA) from the hydrolysis of p-nitro-phenyl acetate to p-nitrophenol.44 The change in absorbance of p-nitrophenol, produced, at 400 nm was measured for 20 min by using Cary-100 UV−vis spectrophotometer. Protein concentration for this experiment was 1.5 M and PNPA concentration was 60 M. The experiments were carried out at 37 C. 3. Results and Discussion 3.1. Circular Dichroism (CD) Spectra and Esterase Activity in Buffer and inside the Gel In Figure 1A, we have represented the circular dichroism (CD) spectra in terms of mean residual ellipticity value MRE 

 ncl  10

, where the concentration (c) is obtained from the

absorbance of the tryptophan residue (inset of Figure 1B) and n denotes the number of amino acids residue = 585 for HSA). Figure 1A shows the CD spectra of unlabeled HSA in a buffer solution and of CPM labeled HSA in buffer solution as well as inside the pluronic F127 gel. The parameters of secondary structure (i.e. alpha helicity, beta sheet, b-turn and random coil) of labeled and unlabeled protein in buffer are given in Table 1. The extent of loss of protein secondary structure (-helix) after labeling is calculated to be 12%. 8


Page 9 of 32

5000

(A)

0

Absorbance

1.00

-5000 HSA_CPM_buffer HSA_buffer HSA_gel HSA_CPM_gel

-10000 -15000 -20000 -25000 200

210

220 230 240 Wavelength (nm)

250

(B)

0.4

Absorbance

2

-1

MRE (deg.cm dmol )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


260

0.75

HSA_CPM_Gel

0.2

HSA_CPM_Buffer

0.1

HSA_Buffer

0.0

0.50 0.25

Only F127

0.3

250

300 350 400 450 Wavelength (nm)

F127 Gel

0.00 200


Figure 1. (A) Circular dichroism (CD) spectra for native HSA in buffer solution (red) as well as inside F127 gel (blue) and CPM labeled HSA in buffer (black) as well as inside the F127 gel (dark cyan). (B) Absorption spectrum of F127 gel, inset shows absorption spectra of the samples corresponding to the CD spectra. F127 gel itself absorbs significantly below ~220 nm. Thus, the CD spectra could not be recorded below 220 nm inside the F127 gel. Table 1. Parameters of Secondary Structure for Labeled and Unlabeled HSA Protein Systems

-helix

Antiparallel -sheet

Parallel sheet

-turn

Random coil

HSA in buffer

65.7

2.9

3.6

11.7

16.1

HSA CPM in buffer

58

4.3

4.4

13.6

19.7

We could not calculate the secondary structure parameters inside the gel as we could not record the CD spectrum below 220 nm (because of the strong absorption of the F127 gel, Figure 1B). From Figure 1A, it is interesting to note that in buffer the ellipticity (MRE) of HSA around 220 nm marginally decreases upon labeling by CPM indicating a slight loss in secondary structure. In F127 gel, the ellipticity of HSA is more than that in buffer. This shows that the crowded environment of the gel stabilizes the secondary structure of the protein. Note, in gel the ellipticity of HSA remains unchanged upon labeling by CPM. In summary, labeling by CPM causes slight loss in secondary structure of the protein in buffer but no change in structure inside 9



the gel. This is consistent with the earlier reports20,21 that molecular crowding enhances stability and structure of the protein. We will show in later that the labeled protein remains biologically active in both buffer and gel. The biological activity (i.e. esterase activity) of the protein (HSA) is performed by monitoring the absorbance of p-nitrophenol (PNP) produced on hydrolysis of p-nitrophenyl acetate.44 As shown in Figure 2, in buffer the biological activity of the labeled protein is slightly lower than that of the unlabeled protein. However, in F127 gel, both labeled and unlabeled proteins show almost similar biological activity. The overall kinetics is slightly slower in the gel presumably because of higher viscosity in the gel.

Absorbance (at 400 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 32

0.10 HSA_PNPA_buffer

0.08 0.06

HSA_CPM_PNPA_buffer

0.04

HSA_CPM_PNPA_gel

0.02

HSA_PNPA_gel

0.00

PNPA_buffer PNPA_gel

0

4

8 12 Time (min)

16

20

Figure 2: Hydrolysis of p-nitrophenyl acetate (PNPA) in presence of unlabeled protein and labeled protein measuring the absorbance of p-nitrophenol (PNP) at 400 nm. 3.2. Steady-state Fluorescence Spectra HSA contains a single tryptophan (Trp-214) residue in domain IIA. Figure 3A represents the fluorescence spectra of HSA from the Trp-214 residue in buffer solution and inside the gel when excited at 295 nm. The heterogeneity in the polarity of the gel leads to broader spectral width (presumably from probes in different environment) of the fluorescence spectrum compared to that in bulk water. CPM dye is non-fluorescent in non-bound state and is highly fluorescent when bound to thiol (-SH) containing group (Scheme 1B). Free CPM is non-fluorescent presumably because of the non-radiative transition from the coumarin moiety to a low lying n-* state of the maleimide 10


Page 11 of 32

ring.45 On addition of a thiol (-SH) to the double bond of the maleimide ring this low lying state is no longer available and hence, the probe becomes highly fluorescent.

Trp Fluorescence

8

HSA_Buffer HSA_Gel

6 4 2 0

350

1.0 (B) 0.8

HSA_CPM

7

1.4x10

6

7.0x10

0.0 420

0.6

Only CPM

450

480

510

540

Wavelength (nm)

0.4 0.2 0.0


7

2.1x10

Fl. Intensity

10 (A)

Normalized Fl. Intensity

5

Fluorescence Intensity [10 ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CPM_HSA_Gel CPM_HSA_Buffer 440

480 520 Wavelength (nm)

560

Figure 3. Fluorescence spectra: (A) tryptophan residue of HSA (Trp-214 residue) in buffer and inside F127 gel. (ex = 295 nm), (B) CPM labeled HSA in buffer and inside F127 gel, inset shows the auto fluorescence of free CPM in bulk solution which is negligible under the experimental conditions (ex = 405 nm). The fluorescence spectra of CPM labeled HSA in buffer and in the pluronic F127 gel are displayed in Figure 3B. The emission maximum of CPM attached to HSA, shows an emission maximum at 460 nm in buffer solution and at 465 nm inside the gel. The 5 nm red shift in the fluorescence maxima implies that the probe CPM, attached to the HSA, is more exposed to bulk (water pool) inside the gel compared to the buffer solution and experiences a more polar local environment. Thus, though the protein HSA is more structured (i.e. more stable) inside the gel as evidenced by CD spectra, different domains (containing tryptophan and CPM) are affected differently. 3.3 FRET from Tryptophan to CPM As shown in Figure 4A, unlabeled HSA exhibits strong tryptophan fluorescence at ~350 nm when excited at λex = 295 nm. In the presence of CPM (i.e. CPM labeled HSA), the intensity of tryptophan (Trp) fluorescence decreases with a concomitant increase in emission at 460 nm, which is assigned to CPM fluorescence. It is evident from the Figure 4A that the contribution of auto-fluorescence coming from the gel as well as free CPM is negligible. 11


18

Page 12 of 32

1000

(A)

Donor (Trp) Lifetime

(B)

6

CPM_HSA_Buffer

15

800

12

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fluorescence Intensity [10 ]


HSA_Buffer

9

Only CPM

6

CPM_HSA_Gel HSA_Gel

400

HSA_Buffer

200

CPM_HSA_Gel

3

CPM_HSA_Buffer

600

IRF

Only F127

0

0 350


550

0

3

6 9 Time (ns)

12

15

Figure 4. (A) Fluorescence spectra: Tryptophan residue of unlabeled HSA in buffer solution (blue); Tryptophan and CPM emission of CPM labeled HSA in buffer solution (red) and in F127 gel (black); Auto fluorescence of F127 (brown) and free CPM dye (green). (B) Fluorescence decays of donor (Trp): unlabeled HSA in buffer solution (brown) as well as inside F127 gel (blue) and CPM labeled HSA in buffer solution (green) as well as inside F127 gel (red) (λex = 295 nm) The decrease in intensity of tryptophan fluorescence and increase in CPM emission suggest fluorescence resonance energy transfer (FRET) from Trp to the CPM attached to the same protein (Figure 4A). FRET is further evidenced by the decrease in the lifetime of donor (tryptophan) for the CPM labeled HSA compared to that in HSA without acceptor (here, CPM) (Figure 4B and Table 2). It is evident that the FRET is less efficient inside the gel compared to that in buffer solution (Figure 4A). Since FRET efficiency (FRET) is inversely proportional to the sixth power of donor - acceptor distance (RDA), this indicates longer distance of tryptophan and CPM inside the gel. We have calculated the FRET efficiency (FRET) using the following equation,46

 τ 1 ε FRET  1  DA  τD  fa 

(8)

where, 𝐷𝐴 and 𝐷 represent the lifetime of the donor (here, Trp-214 residue of the HSA) in presence and absence of acceptor (here, CPM covalently linked to Cys-34 residue), respectively. 12


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We have used average lifetime of the Trp for the calculation of FRET efficiency using the above equation. Labeling efficiency is represented by 𝑓𝑎 (80%, in the present case). For the unlabeled protein, lifetime of the donor, tryptophan is 5.3 ns in buffer and 4.1 ns in F127 gel. So, donor lifetimes of unlabeled protein are found to be different in buffer and gel. According to the above equation, FRET efficiencies are observed to be 50  5% and 25  3% in buffer and gel, respectively. Table 2. Fluorescence Lifetime Decay Parameter for Tryptophan Residue of HSA monitored at em = 350 nm.

System

1 (ns)

2 (ns)

>[a] (ns)

HSA in buffer

3.0 (0.4)

6.8 (0.6)

5.3

HSA in gel

2.4 (0.6)

6.7 (0.4)

4.1

CPM-HSA in buffer

1.2 (0.5)

5.0 (0.5)

3.1

CPM-HSA in F127 gel

2.1 (0.6)

5.2 (0.4)

3.3

[a]

0.2 ns. The distance between the lone tryptophan (Trp-214) to CPM labeled to the Cys-34

residue of HSA i.e. RDA was determined by the FRET experiment. The spectral overlap between the donor (Trp) emission and the acceptor (CPM) absorption is appreciable with an overlap integral, J(λ) = 2.99 X 1014 M-1 cm-1 nm4 (Figure 5A). Using this, Förster radius (R0 value) is calculated to be 3 nm. It may be noted that the CPM covalently attached to HSA/ or cysteine exhibit negligible absorption around 295 nm39,40 as also shown in Figure 5B. The distance (RDA) between the Trp (D) and CPM (A) is calculated using the following relation,46 1

 1   FRET  6 R DA  R 0     FRET 

(9)

13



0.2

(A)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 32

(B)

CPM Absorption

280


0.1

0.0

560

Figure 5. (A) Spectral overlap between emission spectrum of donor (Trp residue of HSA, red, ex = 295 nm) and absorption spectrum of acceptor (CPM, blue) and (B) absorption spectrum of CPM. Thus, we obtained the donor acceptor distances to be 3 ± 0.1 nm and 3.6 ± 0.1 nm in buffer and gel, respectively. Thus, the distance between the Trp and CPM bound to HSA increases nearly 20% in the gel compared to that in the bulk (buffer solution). This leads to lower efficiency of FRET in gel. 3.4

Fluorescence Anisotropy Decay of CPM labeled HSA in Buffer and F127 Gel In this section, we examine the rotational dynamics of the protein (HSA) inside the

crowded environment of the gel using fluorescence anisotropy decay. Fluorescence anisotropy decays of HSA labeled with CPM in bulk (in buffer) and in F127 gel are shown in Figure 6. The decay parameters of rotational dynamics are summarized in Table 3. The high value of time zero anisotropy (r0) suggests that the most of the rotational relaxation is captured in the picosecond setup. Fluorescence anisotropy decays of CPM labeled HSA are observed to be very much slower (>10 ns, Table 3). The fluorescence lifetime of the CPM, covalently linked to HSA, is found to be 3.5 ns. Such a short lifetime of CPM can’t faithfully detect the long component (>10 ns) of fluorescence anisotropy decay of HSA.

14


Page 15 of 32

0.5

(A)

r(t)

0.3

0.3

0.2

0.2

0.1

0.1

3 0 -3

4 0 -4

0

2

4

6 8 10 Time (ns)

0.5

12

(B)

0.4

Res

r(t)

0.5

HSA_CPM in buffer Fitted line

0.4

Res

HSA_CPM in F127 gel Fitted line

0

14

(C)

3

6

9 12 Time (ns)

15

18

Fitted line in buffer Fitted line in F127 gel

0.4 0.3 r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 0.1 0.0

0

3

6 9 Time (ns)

12

Figure 6. Fluorescence anisotropy decay of CPM covalently attached to HSA: (A) in buffer solution, (B) inside the F127 gel; Lower panels show their corresponding residuals, (C) The comparison of the fitted lines in buffer and F127 gel. (ex = 405 nm, em = 460 nm for both the cases). The anisotropy decay for CPM labeled HSA in buffer is fitted to a single exponential with the rotational time-scale of 9 ± 2 ns. Thus, CPM labeled HSA exhibits a major slow component even in bulk solution (buffer). On the other hand, bi-exponential decay of the rotational relaxation is observed inside the gel- a relatively faster component of 0.45 (0.23) ns 15



Page 16 of 32

and a major slow component of 15.7 (0.77) ns. A similar slow time-scale of rotational relaxation (>20 ns) was observed previously for BSA protein in the case of a non-covalently labeled probe (8-anilino-1-naphtalenesulfonate, ANS),47 a Ru(II) complex linked to HSA,48 and for erythrosin bound to BSA.49 In this case, the very slow anisotropy decay arises from the overall tumbling motion of the protein (CPM labeled HSA). Table 3. Parameters of Fluorescence Anisotropy Decay, r(t), for CPM Labeled HSA. System

r0

rot1 (ns)

rot2 (ns)

CPM-HSA in bulk

0.35

-

9.0 (1.0)

CPM-HSA in F127 gel

0.33

0.45 (0.23)

15.7 (0.77)

The rotational relaxation time is proportional to the viscosity of the medium () and the 4 volume ( V  r 3 ) of the rotating group as, 3 τ rot 

ηV kT

(10)

It is evident that the long component of rotational relaxation increases by factor of 1.7 times in the gel compared to bulk water (Table 3). From the FRET experiment, size (r) of the protein increases 20% in the gel compared to bulk water. This corresponds to an increase in volume by 1.7 times (i.e. (1.2)3). Thus, the slightly slower time-scale of rotational dynamics in the gel may be fully accounted for the slight increase in volume where the rotational friction (viscosity) remains more or less unchanged in the immediate environment of the gel. To summarize, the time-scale of rotational relaxation of CPM labeled HSA in F127 gel is close to that in bulk water despite of the 40-300 times higher translational viscosity inside the F127 gel.37 This can be reconciled as follows. According the SANS study, the structure of a F127 gel is close packing structure of the spherical micelles. 50% volume of such a structure is “voids” occupied by water. The diameter of F127 micelles is about 24 nm31-34 and from X-ray crystallography, HSA is triangular molecule (heart shaped protein, Scheme 2A) with each side 16


Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8 nm and thickness about 3 nm.50,51 Thus, a substantial part of the protein is projected outside the micelles into the “void” region of the gel. This indicates that in the crowded environment of the triblock copolymer forming the gel, the protein is located in such a manner so that the probe molecules are more exposed to the bulk aqueous solution (water pool region of the F127 gel, Scheme 2B). As a result, the rotational motion of the protein is similar to that in bulk water in spite of the fact that the viscosity increases 40 times in the corona region and 300 times in the core region inside the gel. This observation is well supported by the 5 nm red shift of the fluorescence maxima of CPM inside the gel compared to that in buffer solution indicating greater exposure of CPM to water inside the gel. In contrast to this, the translational diffusion of the protein is retarded by 59 times inside the gel, will be discussed in the next section. 3.5

Fluorescence Correlation Spectroscopy (FCS) In this section, we use fluorescence correlation spectroscopy (FCS) to determine

translational diffusion and conformational dynamics of the protein in bulk water and in gel. We will show that unlike rotation, translational motion of the protein experience 59 times higher friction (viscosity) in the crowded environment of the gel compared to bulk water. 3.5.1

FCS in Bulk: Translational Diffusion and Size of HSA in Bulk Water FCS traces of CPM labeled HSA in bulk (buffer solution) as well as inside F127 gel are

depicted in Figure 7. The hydrodynamic radius (rH) of CPM labeled HSA is estimated from the value of diffusion coefficient (Dt) using Stoke-Einstein equation (Equation 11) as follows,

Dt 

k BT 6πηrH

(11)

where, , kB and T indicate the viscosity of the system, Boltzmann’s constant and temperature, respectively.

17



(A)

Page 18 of 32

(B)

Cys-34

Trp-214

Scheme 2. (A) Structure of Human Serum Albumin (HSA) showing lone Cys-34 (red) residue located in domain IA and lone Trp-214 (blue) residue in domain IIA (from protein data bank ID: 1A06). The image has been generated using PyMOL Viewer software. (B) A pictorial representation of the location of HSA inside F127 gel. From Figure 7A, it is evident that on going from bulk solution to gel, the magnitude of G(0) decreases. This signifies an increase in the number of fluorophore molecules in the confocal volume. This is due to the change in the viscosity and refractive index mismatch. Chattopadhyay et al.52 have discussed, in detail, how different additives/samples causes change in refractive index and the consequent role of collar setting on focal point and observation volume (i.e. number of fluorophores and G(0)). We have discussed earlier how to take into account of all these factors.54 Briefly, we have corrected for refractive index and viscosity mismatch following the reported standard protocol.52-55 For our purpose, we have used a standard dye, coumarin 102 (C102) as a diffusion standard.41,54 The size (rH) of HSA in buffer solution is obtained through ratio method using the C102, as52-55

rHProtein τ Protein  DC102 C102 rH τD

(12)

where, rH and D correspond to hydrodynamic radius and characteristic diffusion time, respectively. Using Gauss View 5.0.9 software, the size (rH) of C102, is ~0.4 nm.53 The size (rH) of the CPM labeled HSA in bulk solution has been determined using the above equation (Equation 12). 18


Page 19 of 32

Thus, the size (rH) of the HSA is obtained to be 4 nm (diameter ~8 nm) in bulk solution which agrees well with the previously reported size of the protein, HSA.50,51 It may be noted here that the radius (r) of CPM was obtained earlier from theoretical calculation (Gauss View 5.0.9 software) to be 0.95 nm.53 3.5.2

FCS in Gel: Translational Diffusion and Viscosity Sensed by the Probe Covalently

Attached to HSA Protein It can readily be seen from Table 4 that the translational diffusion (i.e. Dt) of CPM labeled to HSA is slowed down by about 59 times in gel compared to that in bulk. Please note that the coefficient for translational diffusion of C102 (coumarin 480) is 600 m2 s-1 in water and 13 m2 s-1 in the F127 gel.37 This indicates a 46 fold increase in viscosity of the gel compared to bulk water. From Table 4, coefficient for translational diffusion for CPM labeled HSA is 59 times smaller than that in buffer (water). Thus according to equation 11, the ratio of hydrodynamic radius of the protein in gel (rG) and in buffer (rB), rG/rB is 59/46= 1.25  0.05. This indicates a 25  5 % increase in radius of the protein inside the gel. This is consistent with 20% increase in donor-acceptor distance (RDA) measured by FRET experiment (Section 3.3).

250 -3

200 150 100

CPM-HSA

(A)

1.0

Buffer F127 Gel

0.8

Normalized G(

300

G() [10 ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

CPM-HSA

0.6

(B) Buffer F127 Gel

0.4 0.2

0 0.0 1E-3 0.01 0.1 1 10 100 1000 1E-3 0.01 0.1 1 10 100 1000 Correlation time (ms) Correlation time (ms)

Figure 7. FCS traces of CPM labeled HSA in buffer and inside F127 gel: (A) Un-normalized and (B) Normalized best fit. (All the FCS data are fitted using Equation 3).

19



3.5.3

Page 20 of 32

Conformational Dynamics of HSA in Buffer Solution and inside the Gel During conformational fluctuation of a protein, when the fluorescent probe (in this case

CPM) comes within one angstrom of an amino group of neighboring residue, electron transfer (ET) quenching takes place. This is the basis of PET-FCS.56-58 In FCS, conformational relaxation is monitored from the fluctuations of fluorescence intensity arising from electron transfer quenching of the fluorescence of the probe by amino group containing residues of the protein.5658

In buffer solution, conformational dynamics of HSA exhibit two components– a fast component of 4 s and a relatively slower component of 95 s (Table 4). The shorter component (4 s) corresponds to segmental motion (chain dynamics) and quenching by residues at close proximity.59,60 The long component (95 s) arises from inter-chain interactions (concerted chain dynamics) and quenching by distant amino groups containing residue within the protein.59,60

Table 4. Diffusion, Viscosity and Conformational Dynamics Obtained from FCS Study System CPM-HSA in buffer CPM-HSA in F127 gel

d (ms)

Dt[a] (m2s-1)

0.37

50

22.00

0.83

Viscosity

R>[b]

R1 (s)

R2 (s)

1

4 (0.15)

95 (0.85)

80

59

220 (0.5)

2000 (0.5)

1100

(cP)

(s)

[a]±10%; [b]±5%.

On going from bulk solution to the gel, the time constant of the faster component increases by 55 times, from 4 s to 220 s. On the other hand, the slower time component is also increased by 20 times magnitude, from 100 s to 2000 s (Table 4). Thus, the average time-scale () of conformational dynamics of HSA is increased by 14 times inside the gel implying the slowing down of the side chain dynamics of the protein. The increase in long component may be attributed to the restriction imposed on the dynamics of the protein by the crowded environment of the gel and steric effects of the polymer chains. This dramatic effect 20


Page 21 of 32

was not considered at all in the previous studies of protein in different gels.27-30 It appears that during translational motion in and out of the gel, the protein, which resides largely in peripheral region projected into the void region, makes periodic excursions inside the forest created by the polymer chains of PEO and PPO inside F127 gel. During this excursion, its conformational dynamics is restricted very significantly by the polymer chains of the gel.

Counts (a.u.)

1.0

CPM HSA in Buffer

(A)

(C)

0.8 0.6 0.4

525 nm 420 nm

0.2 0.0

IRF 0

1.0

3

6 9 Time (ns)

12

15

CPM HSA in F127 Gel (B)

1.0 (D)

0.6 0.4 525 nm

0.2 0.0

Intensity (a.u.)

0.8

Counts (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

6 9 Time (ns)

12

0.8

2500 ps 250 ps 80 ps 0 ps

0.6 0.4 0.2

420 nm 0

t

15

0.0

20000 22000 24000 -1 Wavenumber (cm )

Figure 8. Wavelength dependent picosecond fluorescence transients of CPM labeled HSA: (A) in buffer and (B) inside the F127 gel; TRES: (A) in buffer and (B) inside the F127 gel, Data points denote the actual values and solid lines indicate the best fit. (λex = 405 nm) 3.6. Solvation Dynamics of CPM Labeled HSA in Buffer and inside the F127 Gel We have finally investigated solvent response of CPM covalently attached to the HSA protein in the bulk as well as in gel phase. In bulk water, solvation displays a time-scale of 1 ps. 21


The Journal of Physical Chemistry 61-63

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 32

However, in many constrained environment (proteins, DNA and biological cell), water

exhibits a component of 100-1000 ps.25,26,61-65 Wavelength dependent picosecond fluorescence transients of CPM covalently attached to HSA in buffer as well as inside the gel are shown in Figure 8A,B. The observation of decay at the blue end of the fluorescence spectrum and rise preceding the decay in the red emission wavelength is the clear signature of solvation dynamics occurring in this system.42,43 Time resolved emission spectra (TRES), constructed from the steady-state emission spectra and the best fit of the fluorescence transients, are shown in Figure 8C,D. For CPM labeled HSA in buffer and inside the gel, emission maxima show gradual red shift (Stokes shift) with the passage of time. Emission maxima at time zero i.e. (0) and total dynamic Stokes shift  are evaluated from TRES and tabulated in Table 5. Table 5. Fluorescence Maximum and Parameters Obtained from Decay of Solvent Correlation Function, C(t), for CPM Labeled HSA.

System

Emission maximum

Δνobs[a] [ν0] cm-1

τ1 (a1) ps[b]

τ2 (a2) ps[b]

% missed

[c] ps

CPM HSA in Buffer

460 nm

350 [22000]

150 (0.7)

1800 (0.3)

10

600

CPM HSA in F127 Gel

465 nm

450 [21850]

150 (0.7)

1000 (0.3)

30

300

[a]±

50 cm-1, [b]± 50 ps,

[c]average

solvation time, = a1τ1+a2τ2, ±10%, where τ1 and τ2 are the

individual components with corresponding amplitudes of a1 and a2, respectively.

Decays of solvent correlation function, C(t), for the CPM labeled HSA in buffer solution as well as inside the F127 gel are displayed in Figure 9. The decays are fitted to a bi-exponential function. Decay time components of C(t) for CPM labeled HSA in buffer are found to be ~150 ps (70%) and 1800 ps (30%) with an average solvation time, 600 ps (Table 5). Similarly, the time constants for the decay of C(t) inside the gel are 150 ps (70%) and 1000 ps (30%) with 22


Page 23 of 32

an average solvation time, of 300 ps (Table 5). The percentage missed of solvation (i.e. % missed) in the setup of time resolution 100 ps for CPM labeled HSA are calculated as described earlier by using CPM labeled cysteine40 and given in Table 5. The average solvation time () for CPM labeled HSA is 600 ps in the buffer and inside the gel is 300 ps. Thus, it is observed that the CPM labeled HSA inside the pluronic gel exhibits around 2-fold faster solvation compared to that in the buffer solution (Table 5). The slow components in the solvent relaxation dynamics may be attributed to the motion of the hydrogen bonded water molecules as well as other electrostatic interaction in the protein. It may be mentioned here that the retardation of solvation dynamics is much smaller in magnitude than that the retardation of translational and conformational dynamics.

1.0

CPM HSA

0.8 C(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In Buffer In F127 Gel

0.6 0.4 0.2 0.0 0

700

1400 2100 2800 3500 Time (ps)

Figure 9. Comparison of decay of C(t) for CPM labeled HSA in buffer (blue) and inside the F127 gel (red); Data points denote the actual values of decay and solid lines indicate the best fit.

4. Discussion The main objective of this work is to find out how rotational, translational, conformational and solvation dynamics of a protein are affected in the crowded environment of a hydrogel. The main finding of this work is that the translational and conformational dynamics are affected largely inside the gel, but, the rotational and solvation dynamics are marginally affected.

23



It may be mentioned that using computer simulation, Maroncelli and Fleming predicted that nearly 85% of the solvation dynamics arises from the first solvation shell.66 Thus solvation, by and large, represents the local response of the solvent environment. In bulk water, solvation occurs in  1 ps time scale.61-63 However, in the vicinity of a protein the solvation dynamics exhibit a substantially slower component in 100 ps time scale because of bound to free interconversion.61-65 In the present case, we have detected the slow component of solvation dynamics in 300-600 ps time scale. While solvation dynamics involve solvent motion, rotational and translational dynamics involve motion of the probe solute. Note the rotational dynamics probed by anisotropy decay represent local friction around the probe and hence, reports local viscosity around the probe (CPM) of size about 1 nm. According to wobbling in a cone model, this is perturbed by the overall tumbling of the albumin protein which occurs in 10 ns timescale.40,47 The FCS experiment however, probes a much longer time scale (0.37 milliseconds for labeled HSA in bulk water and 22 milliseconds for CPM labeled HSA in the hydrogel). Note, diffusion occurs over a distance of about 200 nm (/2).56 This corresponds to 22/0.37 59 times increase in translational viscosity inside the gel matrix. In the F127 gel of diameter ~24 nm, the local viscosity varies from 1 cP in the void region (like water) to 300 cP in the hydrophobic core of F127 gel (probed by DCM dye)37 with the viscosity ~18 cP in the corona region (probed by anionic C343 dye) and ~46 cP in the intermediate region probed by C102 dye.37 In this case, the 59 fold slowing down of diffusion arises from motion of HSA labeled with coumarin probe (CPM) in and out of the hydrogel during translation. We now consider the conformational dynamics of the protein inside the crowded hydrogel as well as buffer. The time scale of conformational relaxation (4-100 s  10-5-10-4 s) for CPM labeled HSA is much longer than the time scale of solvation (600 ps  10-9 s) in buffer. HSA is a multi-domain protein.18,19,40,50 Different domains of the proteins are fluctuating with time because of the conformational relaxation of the protein. The conformational fluctuation occurs in a time-scale of microsecond ( = 1000 ns) which is intermediate between that of rotational motion (10 ns) and translational diffusion (millisecond = 1000 microsecond). The conformational relaxation is ascribed to the chain dynamics of the protein.56-60 According to Eaton and co-workers, the fast component of conformational dynamics (in a few s) arises from an adjacent amino containing residue (intra-chain dynamics).59,60 The long component (in 100 s) arises from a distant amino group (inter-chain dynamics). In the present case in bulk water 24


Page 24 of 32

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HSA displays two components of 4 s and 95 s. Inside the gel, the fast component increases ~55-fold to 220 s and the slow component increases ~20 times to 2000 s. Evidently, inside the hydrogel, the chain dynamics of the protein is hindered by the structural constraints imposed by the polymer gel. Thus, we attribute the significant retardation of the conformational dynamics of HSA to the hindrance created by the polymer chains of F127 gel. 5.

Conclusion In this work, we have explored the structure and dynamics of a protein, human serum

albumin (HSA) inside a crowded environment of F127 gel using fluorescence correlation spectroscopy (FCS) and picosecond time-resolved fluorescence spectroscopy. The most intriguing observations of the work are nearly 59 fold decrease in local translational viscosity and nearly 14 fold decrease in average conformational motion of the protein inside the gel compared to the bulk (buffer solution). Compared to this, the rotational viscosity and the solvation dynamics are marginally affected. This may be attributed to the fact that solvation and rotation are both controlled by immediate environment that is more or less similar in the gel and in bulk. As discussed, the CPM labeled probe is primarily located at the corona region of the immobilized micelle with a substantial part projected into the “void” occupied by bulk water. However, translation motion of the CPM labeled protein in and out of the gel is retarded by the polymer chains of F127 gel. The large amplitude motion of the protein during conformational motion and quenching by distant amino groups faces impediment from the forest of polymer chains of the gel. These results may provide a significant contribution on the structures and dynamics of a protein in crowded environment in order to unravel the specific functions in more complicated and heterogeneous system of live biological cell.

Acknowledgement AN and SN are thankful to CSIR, Government of India for awarding fellowships. SC thanks IISER Bhopal for providing fellowship. KB thanks DST JC Bose fellowship for generous research grant. SM thanks INSA, for providing financial support.

25



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Structure, Activity and Dynamics of Human Serum Albumin in a

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